Ezetimibe in Combination With Simvastatin Reduces Remnant Cholesterol Without Affecting Biliary Lipid Concentrations in Gallstone Patients.

Background In randomized trials (SHARP [Study of Heart and Renal Protection], IMPROVE -IT [Improved Reduction of Outcomes: Vytorin Efficacy International Trial]), combination of statin and ezetimibe resulted in additional reduction of cardiovascular events. The reduction was greater in patients with type 2 diabetes mellitus (T2 DM ), where elevated remnant cholesterol and high cardiovascular disease risk is characteristic. To evaluate possible causes behind these results, 40 patients eligible for cholecystectomy, randomized to simvastatin, ezetimibe, combined treatment (simvastatin+ezetimibe), or placebo treatment during 4 weeks before surgery, were studied. Methods and Results Fasting blood samples were taken before treatment start and at the end (just before surgery). Bile samples and liver biopsies were collected during surgery. Hepatic gene expression levels were assessed with qPCR . Lipoprotein, apolipoprotein levels, and content of cholesterol, cholesteryl ester, and triglycerides were measured after lipoprotein fractionation. Lipoprotein subclasses were analyzed by nuclear magnetic resonance. Apolipoprotein affinity for human arterial proteoglycans ( PG ) was measured. Biomarkers of cholesterol biosynthesis and intestinal absorption and bile lipid composition were analyzed using mass spectrometry. Combined treatment caused a statistically significant decrease in plasma remnant particles and apolipoprotein B (ApoB)/lipoprotein content of cholesterol, cholesteryl esters, and triglycerides. All treatments reduced ApoB-lipoprotein PG binding. Simvastatin and combined treatment modified the composition of lipoproteins. Changes in biomarkers of cholesterol synthesis and absorption and bile acid synthesis were as expected. No adverse events were found. Conclusions Combined treatment caused atheroprotective changes on ApoB-lipoproteins, remnant particles, bile components, and in ApoB-lipoprotein affinity for arterial PG . These effects might explain the decrease of cardiovascular events seen in the SHARP and IMPROVE - IT trials. Clinical Trial Registration URL : www.clinicaltrialsregister.eu . Unique identifier: 2006-004839-30).

Combined rifampicin and ursodeoxycholic acid treatment does not amplify rifampicin effects on hepatic detoxification and transport systems in humans.

BACKGROUND: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) were found to stimulate different but complementary hepatobiliary detoxification pathways in gallstone patients. AIM: To study whether single drug effects are sustained or even enhanced by combination of both drugs and whether possible effects are mediated by circulating fibroblast growth factor 19 (FGF19), which has recently been identified as a master regulator of bile acid biosynthesis. METHODS: 20 patients scheduled for laparoscopic cholecystectomy were randomized to a combination of UDCA (1 g/day during 3 weeks before surgery) and RIFA (600 mg/day during 1 week before surgery), or no treatment. Routine biochemistry, lipids, bile acid synthesis (7α-hydroxy-4-cholesten-3-one, C-4) and FGF19 were measured in serum. Bile acids were analyzed in serum and bile. A wedge liver biopsy was taken for determination of expression of hepatobiliary ABC transporters on mRNA and protein levels and of enzymes and regulatory transcription factors involved in the metabolism of biliary compounds on mRNA levels. RESULTS: Combination treatment with both RIFA and UDCA significantly stimulated bile acid and bilirubin detoxification (CYP3A4, p < 0.001), conjugation (UGT1A1, p < 0.001) and elimination (MRP2, p < 0.05), as well as bile acid synthesis (p < 0.05), as compared to untreated controls. Notably, serum FGF19 levels in RIFA- and UDCA-treated patients did not differ from controls. CONCLUSION: Combined treatment with RIFA and UDCA preserves the previously observed beneficial effects of single treatment with RIFA, including stimulation of bile acid synthesis. Most notably, the latter effect in humans is not mediated by FGF19.

Cholesterol synthesis inhibition elicits an integrated molecular response in human livers including decreased ACAT2.

OBJECTIVE: The purpose of this study was to identify how different degrees of cholesterol synthesis inhibition affect human hepatic cholesterol metabolism. METHODS AND RESULTS: Thirty-seven normocholesterolemic gallstone patients randomized to treatment with placebo, 20 mg/d fluvastatin, or 80 mg/d atorvastatin for 4 weeks were studied. Based on serum lathosterol determinations, cholesterol synthesis was reduced by 42% and 70% in the 2 groups receiving statins. VLDL cholesterol was reduced by 20% and 55%. During gallstone surgery, a liver biopsy was obtained and hepatic protein and mRNA expression of rate-limiting steps in cholesterol metabolism were assayed and related to serum lipoproteins. A marked induction of LDL receptors and 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase was positively related to the degree of cholesterol synthesis inhibition (ChSI). The activity, protein, and mRNA for ACAT2 were all reduced during ChSI, as was apoE mRNA. The lowering of HDL cholesterol in response to high ChSI could not be explained by altered expression of the HDL receptor CLA-1, ABCA1, or apoA-I. CONCLUSIONS: Statin treatment reduces ACAT2 activity in human liver and this effect, in combination with a reduced Apo E expression, may contribute to the favorable lowering of VLDL cholesterol seen in addition to the LDL lowering during statin treatment.

Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.

BACKGROUND & AIMS: Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. METHODS: Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RESULTS: RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. CONCLUSIONS: RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Changes in gallbladder bile composition and crystal detection time in morbidly obese subjects after bariatric surgery.

The aim of the present study was to elucidate the mechanisms of development of cholesterol crystals and gallstones during weight reduction in obese subjects. Twenty-five morbidly obese, gallstone-free subjects underwent vertical-banded gastroplasty. Gallbladder bile was collected at the time of the operation via needle aspiration and 1.1-7.3 months after the operation via ultrasound-guided transhepatic puncture of the gallbladder. The mean weight loss was 17 kg. Two patients developed gallstones and 10 patients displayed cholesterol crystals in their bile. In patients with a follow-up time of less than 2 months (n = 13), cholesterol saturation increased from 90% to 114% but tended to decrease in the patients with a follow-up time of more than 2 months. The extraction of the concanavalin-A-binding fraction from gallbladder bile obtained after weight reduction in 7 patients prolonged crystallization detection time from 6 to 10 days. The hexosamine concentration, a marker for mucin, was increased by about 100% in bile obtained in 6 of 7 patients after weight reduction. In conclusion, the results indicate that crystallization-promoting compounds (mucin) are of great importance in the development of cholesterol crystals and gallstones in obese subjects during weight reduction, probably because of defective gallbladder emptying.

Feedback regulation of bile acid synthesis in human liver: importance of HNF-4alpha for regulation of CYP7A1.

A great number of nuclear factors are involved in the negative feedback mechanism regulating bile acid synthesis. There are two major ways for the negative feedback to effect the synthesis; the SHP-dependent, involving FXR, and the SHP-independent way, affecting HNF-4alpha. We studied 23 patients with gallstone disease. Eight patients were treated with chenodeoxycholic acid, 7 with cholestyramine prior to operation, and 8 served as controls. Liver biopsies were analyzed with Real-time-PCR. In the cholestyramine-treated group mRNA levels of CYP7A1 were increased about 10-fold. Treatment with CDCA decreased the mRNA levels of CYP7A1 by about 70%. The mRNA levels of CYP8B1, CYP27A1, and CYP7B1 were not significantly altered in the treated groups. The analysis of mRNA levels for HNF-4alpha showed 64% higher levels in the cholestyramine-treated group compared to the controls. These levels showed positive and highly significant correlation to the levels of mRNA of CYP7A1 when studied in all three groups together. FXR, SHP, and LRH-1/FTF were not significantly affected by the different treatments. Our results indicate that when bile acid synthesis is upregulated by cholestyramine treatment the SHP-independent pathway for controlling CYP7A1 transcription dominates over the SHP-dependent pathway.

ACAT2 is localized to hepatocytes and is the major cholesterol-esterifying enzyme in human liver.

BACKGROUND: Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification. METHODS AND RESULTS: In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined. CONCLUSIONS: The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.

Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor.

BACKGROUND/AIMS: Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance. METHODS: Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis). RESULTS: In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis. CONCLUSIONS: The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.

High level of deoxycholic acid in human bile does not promote cholesterol gallstone formation.

AIM: To study whether patients with excess deoxycholic acid (DCA) differ from those with normal percentage of DCA with respect to biliary lipid composition and cholesterol saturation of gallbladder bile. METHODS: Bile was collected during operation through puncturing into the gallbladder from 122 cholesterol gallstone patients and 46 gallstone-free subjects undergoing cholecystectomy. Clinical data, biliary lipids, bile acid composition, presence of crystals and nucleation time were analyzed. RESULTS: A subgroup of gallstone patients displayed a higher proportion of DCA in bile than gallstone free subjects. By choosing a cut-off level of the 90th percentile, a group of 13 gallstone patients with high DCA levels (mean 50 percent of total bile acids) and a large group of 109 patients with normal DCA levels (mean 21 percent of total bile acids) were obtained. The mean age of the patients with high DCA levels was higher than that of the group with normal levels (mean age: 62 years vs 45 years) and so was the mean BMI (28.3 vs. 24.7). Plasma levels of cholesterol and triglycerides were slightly higher in the DCA excess groups compared with those in the normal DCA group. There was no difference in biliary lipid composition, cholesterol saturation, nucleation time or occurrence of cholesterol crystals in bile between patients with high and normal levels of DCA. CONCLUSION: Gallstone patients with excess DCA were of older age and had higher BMI than patients with normal DCA. The two groups of patients did not differ with respect to biliary lipid composition, cholesterol saturation, nucleation time or occurrence of cholesterol crystals. It is concluded that DCA in bile does not seem to contribute to gallstone formation in cholesterol gallstone patients.

Regulation of hepatic low-density lipoprotein receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol 7alpha-hydroxylase mRNAs in human liver.

To characterize the coordinate regulation of cholesterol metabolism in human liver, we simultaneously quantified mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and low- density lipoprotein receptors (LDLRs) in liver biopsies from 76 patients undergoing cholecystectomy. The three transcript levels were not different between untreated gallstone and gallstone-free patients and not significantly altered by 10-d exclusion of dietary cholesterol. Treatment with chenodeoxycholic acid suppressed CYP7A1 and to a lesser extent HMGR mRNA levels. Cholestyramine treatment increased CYP7A1, but also HMGR and LDLR mRNA, and statins only increased HMGR mRNA. Resin + statin treatment increased all mRNA species. In untreated patients, the mRNA levels of HMGR and LDLR were more strongly correlated (r = +0.60) than those of CYP7A1 and HMGR (r = +0.49) or CYP7A1 and LDLR (r = +0.21). In the treated patients, in whom bile acid synthesis was suppressed or stimulated, mRNA levels of CYP7A1 and HMGR (r = +0.84) as well as CYP7A1 and LDLR (r = +0.62) were more strongly correlated than those of HMGR and LDLR (r = +0.59). The coordinate control of HMGR and LDLR mRNA levels reflects their common regulation by shared transcriptional activation. In contrast, following changes in bile acid flux through the liver, CYP7A1 gene expression becomes a strong modulator of hepatic cholesterol metabolism.